The present invention relates to the use of proteins which are differentially expressed in primary brain tumor tissues, as compared to normal brain tissues, as biomolecular targets for brain tumor treatment therapies. Specifically, the present invention relates to the use of immunotherapeutic and immunoimaging agents which specifically bind to human protein tyrosine phosphatase-zeta (PTP"xgr") for the treatment and visualization of brain tumors in patients. The present invention also provides compounds and pharmaceutically acceptable compositions for administration in the methods of the invention.
Brain Tumor Biology and Etiology
Brain tumors are considered to have one of the least favorable prognoses for long term survival: the average life expectancy of an individual diagnosed with a central nervous system (CNS) tumor is just eight to twelve months. Several unique characteristics of both the brain and its particular types of neoplastic cells create daunting challenges for the complete treatment and management of brain tumors. Among these are 1) the physical characteristics of the intracranial space, 2) the relative biological isolation of the brain from the rest of the body, 3) the relatively essential and irreplaceable nature of the organ mass, and 4) the unique nature of brain tumor cells.
First and foremost, the intracranial space and physical layout of the brain create significant obstacles to treatment and recovery. The brain is made of, primarily, astrocytes (which make up the majority of the brain mass, and serve as a scaffold and support for the neurons), neurons (which carry the actual electrical impulses of the nervous system,) and a minor contingent of other cells such as insulating oligodendrocytes (which produce myelin). These cell types give rise to primary brain tumors (e.g., astrocytomas, neuroblastomas, glioblastomas, oligodendrogliomas, etc.) Although the World Health Organization has recently established standard guidelines, the nomenclature for brain tumors is somewhat imprecise, and the terms astrocytoma and glioblastoma are often used broadly. The brain is encased in the relatively rigid shell of the skull, and is cushioned by the cerebrospinal fluid, much like a fetus in the womb. Because of the relatively small volume of the skull cavity, minor changes in the volume of tissue in the brain can dramatically increase intracranial pressure, causing damage to the entire organ (i.e., xe2x80x9cwater on the brainxe2x80x9d). Thus, even small tumors can have a profound and adverse affect on the brain""s function. In contrast, tumors in the relatively distensible abdomen may reach several pounds in size before the patient experiences adverse symptoms. The cramped physical location of the cranium also makes surgery and treatment of the brain a difficult and delicate procedure. However, because of the dangers of increased intracranial pressure from the tumor, surgery is often the first strategy of attack in treating brain tumors.
In addition to its physical isolation, the brain is chemically and biologically isolated from the rest of the body by the so-called xe2x80x9cBlood-Brain-Barrierxe2x80x9d (or BBB). This physiological phenomenon arises because of the xe2x80x9ctightnessxe2x80x9d of the epithelial cell junctions in the lining of the blood vessels in the brain. Although nutrients, which are actively transported across the cell lining, may reach the brain, other molecules from the bloodstream are excluded. This prevents toxins, viruses, and other potentially dangerous molecules from entering the brain cavity. However, it also prevents therapeutic molecules, including many chemotherapeutic agents that are useful in other types of tumors, from crossing into the brain. Thus, many therapies directed at the brain must be delivered directly into the brain cavity (e.g., by an Ommaya reservoir), or administered in elevated dosages to ensure the diffusion of an effective amount across the BBB.
With the difficulties of administering chemotherapies to the brain, radiotherapy approaches have also been attempted. However, the amount of radiation necessary to completely destroy potential tumor-producing cells also produce unacceptable losses of healthy brain tissue. The retention of patient cognitive function while eliminating the tumor mass is another challenge to brain tumor treatment. Neoplastic brain cells are often pervasive, and travel throughout the entire brain mass. Thus, it is impossible to define a true xe2x80x9ctumor margin,xe2x80x9d unlike, for example, in lung or bladder cancers. Unlike reproductive (ovarian, uterine, testicular, prostate, etc.), breast, kidney, or lung cancers, the entire organ, or even significant portions, cannot be removed to prevent the growth of new tumors. In addition, brain tumors are very heterogeneous, with different cell doubling times, treatment resistances, and other biochemical idiosyncrasies between the various cell populations that make up the tumor. This pervasive and variable nature greatly adds to the difficulty of treating brain tumors while preserving the health and function of normal brain tissue.
Although current surgical methods offer considerably better post-operative life for patients, the current combination therapy methods (surgery, low-dosage radiation, and chemotherapy) have only improved the life expectancy of patients by one month, as compared to the methods of 30 years ago. Without effective agents to prevent the growth of brain tumor cells that are present outside the main tumor mass, the prognosis for these patients cannot be significantly improved. Although some immuno-affinity agents have been proposed and tested for the treatment of brain tumors, see, e.g., the tenascin-targeting agents described in U.S. Pat. No. 5,624,659, these agents have not proven sufficient for the treatment of brain tumors. Thus, therapeutic agents which are directed towards new molecular targets, and are capable of specifically targeting and killing brain tumor cells, are urgently needed for the treatment of brain tumors.
Protein Tyrosine Phosphatase Receptors: Generally, and PTP-Zeta ("xgr")
Vital cellular functions, such as cell proliferation and signal transduction, are regulated in part by the balance between the activities of protein kinases and protein phosphatases. These protein-modifying enzymes add or remove a phosphate group from serine, threonine, or tyrosine residues in specific proteins. Some tyrosine kinases (PTK""s) and phosphatases (PTPase""s) have been theorized to have a role in some types of oncogenesis, which is thought to result from an imbalance in their activities. There are two classes of PTPase molecules: low molecular weight proteins with a single conserved phosphatase domain such as T-cell protein-tyrosine phosphatase (PTPT; MIM 176887), and high molecular weight receptor-linked PTPases with two tandemly repeated and conserved phosphatase domains separated by 56 to 57 amino acids. Examples of this latter group of receptor proteins include: leukocyte-common antigen (PTPRC; MIM 151460) and leukocyte antigen related tyrosine phosphatase (PTPRF; MIM 179590).
Kaplan et al. cloned 3 human receptor PTP genes, including PTP-xcex3 (xe2x80x9cCloning of three human tyrosine phosphatases reveals a multigene family of receptor-linked protein-tyrosine-phosphatases expressed in brain.xe2x80x9d Proc. Nat. Acad. Sci. 87: 7000-7004 (1990).) It was shown that one PTPG allele was lost in 3 of 5 renal carcinoma cell lines and in 5 of 10 lung carcinoma tumor samples tested. PTP-xcex3 mRNA was expressed in kidney cell lines and lung cell lines but not in several hematopoietic cell lines tested. Thus, the PTP-xcex3 gene appeared to have characteristics suggesting that it may be a tumor suppressor gene in renal and lung carcinoma. Bamea et al. (xe2x80x9cIdentification of a carbonic anhydrase-like domain in the extracellular region of RPTP-gamma defines a new subfamily of receptor tyrosine phosphatases.xe2x80x9d Molec. Cell. Biol. 13: 1497-1506 (1993)) cloned cDNAs for the human and mouse PTP-xcex3 gene (designated PTP-xcex3 by that group) from brain cDNA libraries, and analyzed their predicted polypeptide sequences. The human (1,445-amino acid) and mouse (1,442-amino acid) sequences share 95% identity at the amino acid level and predict a putative extracellular domain, a single transmembrane domain, and a cytoplasmic region with 2 tandem catalytic tyrosine phosphatase domains. The extracellular domain contains a stretch of 266 amino acids that are highly similar to the zinc-containing enzyme carbonic anhydrase (MIM 114800), suggesting that PTP-xcex3 and PTP"xgr" represent a subfamily of receptor tyrosine phosphatases. The gene for PTP-xcex3 has 30 exons and is approximately 780 kb in size. It is much larger than the other receptor PTP genes, with the CD45 gene (MIM 151460) being around 100 kb and the others even smaller.
Another receptor-type tyrosine phosphatase, protein tyrosine phosphatase zeta (PTP"xgr") [also known as PTPRZ, HPTP-ZETA, HPTPZ, RPTP-BETA(xcex2), or RPTPB] was isolated as a cDNA sequence by two groups in the early nineties. The complete cDNA sequence of the protein is provided in SEQ ID NO. 1, and the complete deduced amino acid sequence is provided in SEQ ID NO. 2. Splicing variants and features are indicated in the sequences. Levy et al. (xe2x80x9cThe cloning of a receptor-type protein tyrosine phosphatase expressed in the central nervous systemxe2x80x9d J. Biol. Chem. 268: 10573-10581, (1993)) isolated cDNA clones from a human infant brain step mRNA expression library, and deduced the complete amino acid sequence of a large receptor-type protein tyrosine phosphatase containing 2,307 amino acids.
Levy found that the protein, which they designated PTP-xcex2 (PTP"xgr"), is a transmembrane protein with 2 cytoplasmic PTPase domains and a 1,616-amino acid extracellular domain. As in PTP-xcex3 (MIM 176886), the 266 N-terninal residues of the extracellular domain are have a high degree of similarity to carbonic anhydrases (see MIM 114880). The human gene encoding PTP"xgr" has been mapped to chromosome 7q31.3-q32 by chromosomal in situ hybridization (Ariyama et al., xe2x80x9cAssignment of the human protein tyrosine phosphatase, receptor-type, zeta (PTPRZ) gene to chromosome band 7q31.3xe2x80x9d Cytogenet. Cell Genet. 70: 52-54 (1995)). Northern blot analysis has shown that showed that PTP-zeta is expressed only in the human central nervous system. By in situ hybridization, Levy et al. (1993) localized the expression to different regions of the adult human brain, including the Purkinje cell layer of the cerebellum, the dentate gyrus, and the subependymal layer of the anterior horn of the lateral ventricle. Levy stated that this was the first mammalian tyrosine phosphatase whose expression is restricted to the nervous system. In addition, high levels of expression in the murine embryonic brain were said to suggest an important role in CNS development.
Gebbink et al. isolated a mouse cDNA of 5.7 kb, encoding a xe2x80x98newxe2x80x99 member of the family of receptor-like protein-tyrosine phosphatases, termed RPTP-xcexc (xe2x80x9cCloning, expression and chromosomal localization of a new putative receptor-like protein tyrosine phosphatase.xe2x80x9d FEBS Lett. 290: 123-130 (1991)). The cDNA predicted a protein of 1,432 amino acids (not including the signal peptide) with a calculated molecular mass of 161,636. In addition, they cloned the human homolog, which showed 98.7% amino acid identity to the mouse protein. The predicted mouse protein consisted of a 722-amino acid extracellular region, containing 13 potential N-glycosylation sites, a single transmembrane domain, and a 688-amino acid intracellular part containing two tandem repeats homologous to the catalytic domains of other tyrosine phosphatases. RNA blot analysis showed a single transcript that was most abundant in lung but present in much lower amounts in brain and heart as well. The human PTP-xcexc gene was assigned to 18pter-q11 by Southern analysis of human/rodent somatic cell hybrid clones.
PTP-xcex5 cDNA was isolated by Krueger et al. (Structural diversity and evolution of human receptor-like protein tyrosine phosphatases. EMBO J. 9:3241-3252, 1990.1990). The 700-amino acid protein has a short extracellular domain and two tandemly repeated intracellular PTPase domains. High levels of PTP-xcex5 transcription were noted in the mouse brain and testes. Both iso forms of PTP-xcex5xe2x80x94a transmembrane, receptor-type isoform and a shorter, cytoplasmic onexe2x80x94appear to arise from a single gene through the use of alternative promoters and 5-prime exons.
Thus, the PTP receptor family of proteins has been characterized as a fairly diverse family of membrane-bound receptors, and non-membrane bound isoforms, which share a common PTPase cytosol domain architecture. Although their expression in fetal and embryonic tissues has suggested a developmental biology role for the proteins, their full function in normal and disease state biology is still not fully understood.
The present invention provides novel methods and reagents for specifically targeting brain tumor neoplastic cells for both therapeutic and imaging purposes. Thus, in a first aspect, the present invention provides PTP"xgr" affinity-based compounds and compositions useful in treating a brain tumor in a patient. The compositions and compounds of this aspect of the invention generally fall into two groups: PTP"xgr"-binding conjugate compounds, which comprise a cytotoxic moiety (C), which inhibits the growth of tumor cells; and PTP"xgr"-binding compound compositions in which the PTP"xgr" binding moiety alters the normal function of PTP"xgr" in the tumor cell, thus inhibiting cell growth.
In a first group of embodiments of this aspect of the invention, PTP"xgr"-binding therapeutic conjugate compounds are provided. These compounds have the general formula xcex1(Pz)C, wherein xcex1(Pz) is one or more moieties which specifically binds to a human protein tyrosine phosphatase-zeta, and C is one or more cytotoxic moieties. In preferred embodiments xcex1(Pz) is an antibody or an antibody fragment. In particularly preferred embodiments, xcex1(Pz) is an antibody or an antibody fragment which elicits a reduced immune response when administered to a human patient. Preferred cytotoxic moieties for use in these embodiments of the invention include radioactive moieties, chemotoxic moieties, and toxin proteins. The invention also provides compositions comprising these PTP"xgr"-binding therapeutic conjugate compounds in a pharmaceutically acceptable carrier.
In a second group of embodiments of this first aspect of the invention, PTP"xgr"-binding therapeutic compounds are provided which alter the normal function of PTP"xgr" in brain tumor cells and inhibit brain tumor cell growth. These PTP"xgr"-binding therapeutic compounds have the general formula xcex1(Pz), wherein xcex1(Pz) is one or more moieties which specifically binds to a human protein tyrosine phosphatase-zeta, and wherein the binding of xcex1(Pz) alters the function of protein tyrosine phosphatase-zeta. In preferred embodiments xcex1(Pz) is an antibody or an antibody fragment. In particularly preferred embodiments, xcex1(Pz) is an antibody or an antibody fragment which elicits a reduced immune response when administered to a human patient. It is preferred that the therapeutic compounds of this second group of embodiments of the first aspect of the invention be formulated into therapeutic compositions comprising the PTP"xgr"-binding compound in a pharmaceutically acceptable carrier.
In a second aspect, the present invention provides methods for using these compounds and compositions to treat a brain tumor in a patient. The methods comprise administering an effective amount of a composition, comprising a PTP"xgr"-binding compound from the first or second group of embodiments of the first aspect and a pharmaceutically acceptable carrier, to a patient in need thereof. Brain tumors treated in this fashion may be glioblastomas, astrocytomas, neuroblastomas, or any type of brain tumor. Administration of the therapeutic composition may be by any acceptable means. One preferred method for administration is by intrathecal administration, although intravascular administration is also preferred.
In a third aspect, the present invention provides PTP"xgr" affinity-based compounds and compositions for the visualization of brain tumors in patients. These compounds have the general formula xcex1(Pz)I, wherein xcex1(Pz) is one or more moieties which specifically binds to a human protein tyrosine phosphatase-zeta, and I is one or more imaging moieties. In preferred embodiments xcex1(Pz) is an antibody or an antibody fragment. In particularly preferred embodiments, xcex1(Pz) is an antibody or an antibody fragment which elicits a reduced immune response when administered to a human patient. Preferred I moieties include radiographic moieties (useful in, e.g., x-ray, scintillation, or other radiation imaging methods,) positron-emitting moieties, magnetic spin contrast moieties, and optically visible moieties (such as visible particles, fluorescent dyes, and visible-spectrum dyes.) It is preferred that the imaging compounds of these embodiments of the third aspect of the invention be formulated into therapeutic compositions comprising the PTP"xgr"-binding compound in a pharmaceutically acceptable carrier.
In a fourth aspect, the present invention provides methods of using the compounds and compositions of the third aspect of the invention to visualize a brain tumor in a patient. These methods generally comprise administering an effective amount of an imaging compound of the general formula xcex1(Pz)I in a pharmaceutically acceptable carrier to the patient, and then visualizing the imaging moieties of the compound. Administration of the imaging composition may be by any acceptable means. Intravascular administration of the imaging composition is preferred in these methods, although intrathecal administration is also preferred. Preferred methods of visualizing the imaging moieties of the compounds include radiographic imaging techniques (e.g., x-ray imaging and scintillation imaging techniques), positron-emission tomography, magnetic resonance imaging techniques, and direct or indirect (e.g., endoscopic) visual inspection.